Original Article Biomol Ther 21(2), 132-137 (2013) Protective Effects of Geniposide and Genipin against Hepatic Ischemia/Reperfusion Injury in Mice Joonki Kim1, Hyo-Yeon Kim2 and Sun-Mee Lee2,* 1Natural Medicine Center, Korea Institute of Science & Technology, Gangneung 210-340, 2School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea Abstract Geniposide is an active product extracted from the gardenia fruit, and is one of the most widely used herbal preparations for liver disorders. This study examined the cytoprotective properties of geniposide and its metabolite, genipin, against hepatic ischemia/ reperfusion (I/R) injury. C57BL/6 mice were subjected to 60 min of ischemia followed by 6 h of reperfusion. Geniposide (100 mg/ kg) and genipin (50 mg/kg) were administered orally 30 min before ischemia. In the I/R mice, the levels of serum alanine amino- transferase and hepatic lipid peroxidation were elevated, whereas hepatic glutathione/glutathione disulfide ratio was decreased. These changes were attenuated by geniposide and genipin administration. On the other hand, increased hepatic heme oxygen- ase-1 protein expression was potentiated by geniposide and genipin administration. The increased levels of tBid, cytochrome c protein expression and caspase-3 activity were attenuated by geniposide and genipin. Increased apoptotic cells in the I/R mice were also significantly reduced by geniposide and genipin treatment. Our results suggest that geniposide and genipin offer signifi- cant hepatoprotection against I/R injury by reducing oxidative stress and apoptosis. Key Words: Geniposide, Genipin, Ischemia, Reperfusion, Liver, Apoptosis INTRODUCTION 2004). Oral administration of geniposide increased hepatic glutathione (GSH) content, which is responsible for hepatopro- Ischemia/reperfusion (I/R) injury to the liver is of clinical im- tection against aflatoxin B1-induced liver injury in rats (Kanget portance in humans after hemorrhagic and cardiogenic shock, al., 1997). Furthermore, geniposide protected against hepatic liver surgery and liver transplantation (Sasaki et al., 1997). Al- steatosis in rats fed a high fat diet through its anti-oxidant ac- though the nature of I/R has been widely studied, the mecha- tions and its regulation of adipocytokine release (Ma et al., nisms by which organ damage occurs are unclear. The initial 2011). Genipin, an aglycon of geniposide and a major compo- hepatic I/R injury is known to be triggered by reactive oxygen nent of gardenia fruit, not only demonstrated remarkable anti- species (ROS) with inflammation involving chemokines and inflammatory and anti-angiogenesis effects, but also inhibited cytokines, followed by neutrophil-mediated hepatic injury oc- lipid peroxidation and the production of nitric oxide (Koo et curring in the late period of reperfusion (Jaeschke, 2006). Re- al., 2004b). Given that geniposide is hydrolyzed to genipin by cent evidence has shown that ROS can also induce apopto- intestinal bacteria (Akao et al., 1994), a detailed comparison sis, which is a mechanism for cell death following reperfusion of pharmacological activities of geniposide and genipin would of the ischemic liver (Yu et al., 2011). be useful. The fruit of Gardenia jasminoides Ellis (Rubiaceae) is a tra- Therefore, we examined the protective effects of genipo- ditional Chinese medicinal source which has long been used side and genipin against I/R-induced hepatic injury, particu- for the treatment of inflammation, jaundice and hepatic disor- larly on the oxidative stress and apoptosis. ders (Xu et al., 2008). Geniposide is one of the major iridoid glycosides of gardenia fruit. Geniposide has been shown to possess anti-inflammatory, anti-oxidant, anti-carcinogenic and anti-angiogenic activities (Liaw and Chao, 2001; Kuo et al., Open Access http://dx.doi.org/10.4062/biomolther.2013.005 Received Jan 14, 2013 Revised Feb 8, 2013 Accepted Feb 13, 2013 This is an Open Access article distributed under the terms of the Creative Com- *Corresponding Author mons Attribution Non-Commercial License (http://creativecommons.org/licens- E-mail: [email protected] es/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, Tel: +82-31-290-7712, Fax: +82-31-292-8800 and reproduction in any medium, provided the original work is properly cited. Copyright © 2013 The Korean Society of Applied Pharmacology www.biomolther.org 132 Kim et al. Geniposide & Genipin in Hepatic Ischemia/Reperfusion MATERIALS AND METHODS 5,5’-dithio-bis(2-nitrobenzoic acid), and NADPH, at a wave- length of 412 nm (Tietze, 1969). The oxidized GSH (GSSG) Hepatic I/R procedure level was measured by the same method in the presence of Male C57BL/6 mice weighing 23-25 g (Dae Han Biolink 2-vinylpyridine, and the GSH/GSSG ratio was calculated. Co., Ltd., Eum-sung, Korea) were fasted for 18 h before the experiments but were provided with tap water ad libitum. All Western blot analysis experiments were approved by the Animal Care Committee Cytosolic proteins were isolated from mice liver tissues us- of Sungkyunkwan University and performed in accordance ing NE-PER® Nuclear and Cytoplasmic Extraction Reagents with the guidelines of the National Institute of Health. Mice (Pierce Biotechnology, Rockford, IL, USA) according to the were anesthetized intraperitoneally with ketamine (55 mg/kg manufacturer’s instructions. A 20 μg sample of protein from body weight) and xylazine (7 mg/kg body weight), while body each liver homogenate of cytosolic fraction was loaded per temperature was maintained at 37°C throughout the anesthe- lane on various percent polyacrylamide gels for electrophore- sia using heating pads. A transverse incision was made to the sis. The proteins were then transferred to nitrocellulose mem- abdomen, and the left branches of the portal vein and hepatic branes and incubated at room temperature for 60 min in a buf- artery were clamped with a micro-serrefine clip (Fine Science fer solution containing 5% dried skim milk to block non-specific Tools Inc., Vancouver, BC, Canada) to induce complete isch- binding. Bands were immunologically detected using poly- emia of the median and left lobes of the liver. The right lobes clonal antibodies against mouse heme oxygenase-1 (HO-1) remained perfused to prevent venous congestion of the in- (Cell Signaling Technology Inc., Beverly., MA, USA; 1:1,000), testine. After 60 min of ischemia, the clamp was removed to truncated BH3 interacting domain death agonist (tBid) (Cell allow reperfusion. The wound was closed in layers using silk Signaling Technology Inc.; 1:1,000), cytochrome c (Cell Sig- sutures and the animals were returned to their cages and al- naling Technology Inc.; 1:1,000) and monoclonal antibody lowed to recover from the anesthesia. The sham-operated ani- against mouse β-actin (Sigma-Aldrich; 1:2,500). The binding mals underwent the same procedure but without vessel occlu- of all antibodies was detected using an ECL detection system sion. The mice were sacrificed 6 h after reperfusion, and blood (iNtRON Biotechnology Co., Ltd., Sungnam, Korea) according samples and ischemic liver tissues were collected. The liver to the manufacturer’s instruction. The visualized immunoreac- tissues were immediately analyzed for histological changes tive bands were evaluated densitometrically with ImageQuant (aliquots from the left lobe) and blood was examined for serum TL software (Amersham Biosciences/GE Healthcare Life Sci- alanine aminotransferase (ALT) and aspartate aminotransfer- ence, Piscataway, NJ, USA). ase (AST) activities. The remaining major portions of the liver tissues were immediately frozen in liquid nitrogen and kept at Histological analysis -75°C for later analysis. Liver tissues were fixed immediately in 10% neutral buff- ered formalin, embedded in paraffin and cut serially into 5 μm Administration of geniposide and genipin sections. For apoptotic cell detection, terminal deoxynucleo- Geniposide (ChromaDex Co., Irvine, CA, USA), dissolved tidyl transferase-mediated dUTP nick end-labeling (TUNEL) in saline, and genipin (Wako Pure Chemical Industries., Ltd., staining, which labels the characteristic DNA double strand Osaka, Japan), dissolved in 10% Tween 80 saline, were ad- breaks, was performed using a commercially available kit (In ministered orally at 100 and 50 mg/kg, respectively, 30 min situ Apoptosis Detection Kit; TaKaRa Co., Shiga, Japan). Un- before the sustained ischemia. The dose and timing of genipo- der optical microscopy (OLYMPUS OPTICAL Co., Tokyo, Ja- side and genipin treatments were selected based on previous pan), the number of TUNEL-positive cells in ×400 histological reports (Koo et al., 2006) and our preliminary experiments. fields were counted per liver section. Mice were divided into six treatment groups: (1) vehicle-treat- ed sham, (2) geniposide-treated sham, (3) genipin-treated Caspase-3 activity sham, (4) vehicle-treated I/R, (5) geniposide-treated I/R, and Caspase-3 activity was measured using a fluorogenic pep- (6) genipin-treated I/R. Since there were no differences in any tide substrate, N-acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoro- of the parameters among the three sham groups, groups (1), methyl-cumarine (DEVD-AFC; Bio-Mol, Plymouth Meeting, (2) and (3) were pooled and are referred to as the sham group. PA, USA), according to a previous procedure (Morin et al., 2004). A 30 μg sample of the liver cytosolic protein was incu- Serum aminotransferase activity bated in a buffer containing 30 mM N-[2-hydroxyethyl] pipera- The serum ALT and AST activities were determined by zine-N’-[2-ethanesulfonic acid] (HEPES), 0.3 mM EDTA, 100 standard spectrophotometric procedures using ChemiLab mM NaCl, 0.15% Triton X-100 and 10 mM dithiothreitol. The ALT and AST assay kits (IVDLab Co., Ltd., Uiwang, Korea), samples were incubated at room temperature for 15 min. The respectively. caspase reaction was then initiated by adding 200 μM DEVD- AFC, and the resulting mixture was incubated at 37°C. The Hepatic lipid peroxidation and glutathione content change in fluorescence (excitation at 400 nm and emission at The steady-state level of malondialdehyde (MDA), which is 490 nm) was monitored after 120 min.